Routine maintenance of cell suspensions by repeated weekly subculture is labor intensive and creates cell cultures that change over time. Cryopreservation of cells addresses some of these issues. For example, U.S. Pat. Nos. 5,965,438; 6,127,181; and 6,753,182 relate to some techniques for cryopreservation of plant cells.
Sarkar et al. (Cryobiology 47 [2003] 44-58) relates to defining mechanistic pathways involved in cryopreservation-induced damage of CD4+ T-cells, and to evaluating a cytokine treatment of the cryopreserved samples to rescue apoptosis for the potential future use of the cryopreserved peripheral blood mononuclear cells (PBMC). Using cryopreserved PBMC samples isolated from naïve and Simian immunodeficiency virus (SIV)-infected rhesus macaques, Sarkar et al. report that frozen PBMC showed significantly increased levels of apoptosis-induced CD4+ T-cell death compared to fresh PBMC over a 5-day culture period. Sarkar et al. report that mechanistic studies using a broad-spectrum caspase inhibitor (z-VAD) demonstrated an involvement of caspases in cryopreservation-induced apoptosis of CD4+ T-cells. Sarkar et al. evaluated the ability of a combined IL-2, IL-4, and IL-7 cytokine treatment of the cryopreserved cells to rescue apoptosis of the CD4+ T-cells. Sarkar et al. reported that efficient rescue of cryopreserved CD4+ T-cells has clinical significance in immune function analysis of longitudinal samples and in various long-term protocols requiring cryopreservation, including bone marrow and stem cell transplantation.
In an effort to reduce labor as well as culture variation, cryopreservation of non-transformed and transformed plant cultures and master seed stocks were developed and optimized as described in WO 2006/052835 and US 2006-0101539. A master seed stock may be utilized as a primary source of starting material for the generation of transgenic product or as a primary starting source of transformed plant cells for manufacturing biopharmaceuticals. Consistent recover of plant cultures and master seed stocks over time often deteriorates and may result in no actively growing cells from the master seed stock. Even thought immediate post thaw viability may be excellent as visualized with FDA stain, 24 hours post thaw cultures often show substantial DNA degradation accompanied by damage and cell death.
To date, there has been no illustration of techniques for rescuing plant cell cultures and master seed stocks from cryopreservation-induced damage.